Esterification products of salicylic acid and C13-(branched) alcohols

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28/11/2017 to 12/2/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

17 July 1992

Deviations:

no

Principles of method if other than guideline:

In addition, the following ISO standard was followed:
ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium", (1995).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tridecyl 2-hydroxybenzoate

EC Number:

947-975-7

Molecular formula:

C20H32O3

IUPAC Name:

tridecyl 2-hydroxybenzoate

Specific details on test material used for the study:

Test item information
Identification: Dermol TDSA
Appearance: Colourless to pale yellow clear liquid
Batch: P7202
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 11 November 2021 (expiry date)

Additional information
Test Facility test item number: 209078/A
Purity/Composition correction factor: No correction factor required
Organic carbon (wt%): 75.1%
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Tridecyl Salicylate

CAS number: 19666-16-1
Molecular structure: Not indicated
Molecular formula: C20H32O3
Molecular weight: 320
Highly reactive to water: Not indicated
Volatile: Not indicated
Solubility in water: Insoluble
Stability in water: Stable

Study design

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

-Source
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
-Treatment
The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 4 g/L in the concentrated sludge. The magnetically stirred sludge was used as inoculum at the amount of 15 mL/L of mineral medium, leading to a concentration SS of 30 mg/L.
-Reason for selection
The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 16 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

ca. 12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Reference Item Concentration and Preparation of Test Solutions

A solution of sodium acetate was prepared by dissolving 799.41 mg in Milli- RO water and making this up to a total volume of 200 mL. Volumes of 20 mL from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/L).

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis or equivalent document was provided to the Test Facility and is presented in Appendix 3.

Reserve Samples
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

Test Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Test Concentration and Preparation of Test Solutions
Dermol TDSA was a colourless to pale yellow clear liquid (UVCB). The test item was tested in duplicate at a target concentration of 16 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.
Since Dermol TDSA was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 31.56 mg; test item bottle B: 32.31 mg and toxicity control bottle: 31.69 mg). To this end, weighed amounts were added to watch glasses, which were added directly to the test bottles. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Any residual volumes were discarded.

TEST SYSTEM

Source
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment
The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 4 g/L in the concentrated sludge. The magnetically stirred sludge was used as inoculum at the amount of 15 mL/L of mineral medium, leading to a concentration SS of 30 mg/L.

Reason for selection
The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Testing Strategy and Experimental Design

Test Procedure and Conditions

Test duration
28 days for the inoculum blank and test item (last CO2 measurement on day 29).
14 days for the positive and toxicity control (last CO2 measurement on day 15).
During the test period, the test media were aerated and stirred continuously.

Test vessels
2 litre brown coloured glass bottles.

Milli- RO water
Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.

Stock solutions of mineral components
A)8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2
B)22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.

Mineral medium
1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.

Barium hydroxide
0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm)
A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination
The test media were excluded from light.

Preparation of Bottles

Pre-incubation medium
The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles).
Positive control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).

Preparation
At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Determination of CO2
Experimental CO2 production
The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).

Measurements
Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (positive and toxicity control) and on day 29 (remaining vessels).

Theoretical CO2 production
The theoretical CO2 production was calculated from the molecular formula.

Measurements and Recordings
pH At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.
Temperature of medium Continuously in a vessel with Milli- RO water in the same room.

INTERPRETATION
Acceptability of the Test
1. The reference item was biodegraded by at least 60% (80%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 16%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (79.7 mg CO2 per 2 litres of medium, corresponding to 39.9 mg CO2/L).
4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

ANALYSIS
ThCO2, expressed as mg CO2/mg test item, was calculated as follows:

ThCO2 = ( No. of carbon atoms in test item × Molecular weight CO2 ) / Molecular weight of test item

The first step in calculating the amount of CO2 produced is to correct for background (endogenous) CO2 production. Thus the amount of CO2 produced by a test item is determined by the difference (in mL of titrant) between the experimental and blank Ba(OH)2 traps.
The amount of 0.05 M HCl titrated is converted into mg of CO2 produced:

mg CO2 = ( 0.05 × ∆ mL HCl titrated ) / 2 × 44 = 1.1 × ∆ mL HCl titrated

Relative biodegradation values were calculated from the cumulative CO2 production relative to the ThCO2. A figure of more than 10% biodegradation was considered biologically relevant.
The relative biodegradation values were plotted versus time together with the relative biodegradation of the positive control. Assessment of ready biodegradability was made based on the average biodegradation in test item bottle A and B. If applicable, the number of days was calculated from the attainment of 10% biodegradation until 60% biodegradation. If this period was ≤ 10 days (10-day window) the test item was designated as readily biodegradable.
Toxicity control: if less than 25% biodegradation (based on the combined ThCO2 of the test item and reference item) occurred within 14 days, the test item was assumed to be inhibitory.
The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in mL of titrant) between the blank Ba(OH)2 traps and untreated Ba(OH)2 (background).

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

ca. 10

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

ca. 25

Sampling time:

28 d

Any other information on results incl. tables

Theoretical CO₂Production

The ThCO₂of Dermol TDSA was calculated to be 2.75 mg CO₂/mg.

The ThCO₂of sodium acetate was calculated to be 1.07 mg CO₂/mg.

If applicable the ThCO₂per test bottle are given in the subscript of the tables (seeAppendix 1).

Biodegradation

All data are presented inAppendix 1. The results of CO₂production and biodegradation in blank bottles, background bottles and each test bottle are listed inTable 2to 8.

Table 9 contains the comparison of biodegradation of Dermol TDSA in bottles A and B. 

Figure 1 (attached) shows the curves for biodegradation of the two bottles with Dermol TDSA, the positive control and the toxicity control.

 

The relative biodegradation values calculated from the measurements performed during the test period revealed 25% and 10% biodegradation of Dermol TDSA, for A and B, respectively (based on ThCO₂).

However, average biodegradation of Dermol TDSA in bottle A and B did not reach ≥60% within a 10-day window. Thus, the criterion for ready biodegradability was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 days (45%, based on ThCO₂). Therefore, the test item was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve (see also paragraph 5.1).

            

Monitoring of Temperature and pH

The temperature recorded in a vessel with water in the same room varied between 22 and 23°C. The pH values of the different test media are presented inTable 1.

Table 1         pH Values of Different Test Media

Test medium:	At the start of the test:	On day 14:	On day 28:
Blank control (A)	7.5	-	7.5
Blank control (B)	7.5	-	7.5
Positive control	7.6	7.7	-
Dermol TDSA (A)	7.6	-	7.5
Dermol TDSA (B)	7.6	-	7.5
Toxicity control	7.6	7.6	-

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In conclusion, Dermol TDSA was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The objective of the study was to evaluate the test item Dermol TDSA for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge; Carbon dioxide (CO₂) evolution test (modified Sturm test).

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the ISO standard 10634, 1995.

Dermol TDSA was a colourless to pale yellow clear liquid (UVCB). The test item was tested in duplicate at a target concentration of 16 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical CO₂production (ThCO₂) of Dermol TDSA was calculated to be 2.75 mg CO₂/mg.

The study consisted of six bottles:

2 inoculum blanks (no test item), 

2 test bottles (Dermol TDSA), 

1 positive control (sodium acetate) and 

1 toxicity control (Dermol TDSA plus sodium acetate).

    

Since Dermol TDSA was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, weighed amounts were added to watch glasses, which were added directly to the test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days for the inoculum blank and test item (last CO₂measurement on day 29) and 14 days for the positive and toxicity control (last CO₂measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed 25% and 10% biodegradation of Dermol TDSA, for A and B, respectively (based on ThCO₂).

However, average biodegradation of Dermol TDSA in bottle A and B did not reach ≥60% within a 10-day window. Thus, the criterion for ready biodegradability was not met.

In the toxicity control, Dermol TDSA was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, Dermol TDSA was designated as not readily biodegradable.